LOCUS       sequence-417793-        7379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV genome backbone for AAV-Perturb-seq with direct gRNA capture and
            nuclei sorting based on eGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7379)
  AUTHORS   Santinha AJ, Klingler E, Kuhn M, Farouni R, Lagler S, Kalamakis G, 
            Lischetti U, Jabaudon D, Platt RJ
  TITLE     Transcriptional linkage analysis with in vivo AAV-Perturb-seq.
  JOURNAL   Nature. 2023 Oct;622(7982):367-375. doi: 10.1038/s41586-023-06570-y.
            Epub 2023 Sep 20.
  PUBMED    37730998
REFERENCE   2  (bases 1 to 7379)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-023-06570-y"; journalName: "Nature"; date: 
            "2023-10"; volume: "622"; issue: "7982"; pages: "367-375"
COMMENT     Sequence Label: pAS088
FEATURES             Location/Qualifiers
     source          1..7379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     179..200
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     473..495
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(533..551)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        619..723
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(724..792,793..1584)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(942..961)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1755..2343
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2244..2263
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2405..2534
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2549..2797
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2549..2569
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2720..2739
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     3134..3156
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3148..3165
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3149..3165
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        3188..3263
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        3282..3567
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          3804..4031
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             4052..4072
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5099..5125
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5141..5194
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5195..5308
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             join(5360..5362,5363..6073)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5360..5362,5363..6073)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5402..5423)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5663..5682)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6010..6031
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    6371..6959
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6424..6444)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6842..6853)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   7046..7186
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7046..7175
                     /label=AAV2 ITR
     rep_origin      join(7261..7379,1..337)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7348..7367)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
       61 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
      121 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
      181 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
      241 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
      301 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
      361 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
      421 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgacctt ctccgggagc
      481 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgatacgaaa gggcctcgtg
      541 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
      601 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
      661 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
      721 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
      781 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
      841 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
      901 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
      961 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     1021 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     1081 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     1141 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     1201 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     1261 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     1321 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     1381 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     1441 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     1501 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     1561 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     1621 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     1681 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1741 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1801 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1861 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     1921 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1981 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2041 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2101 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2161 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2221 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     2281 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     2341 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     2401 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
     2461 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     2521 cactaggggt tcctgcggcc gcacgcgtga gggcctattt cccatgattc cttcatattt
     2581 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     2641 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     2701 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     2761 cttggcttta tatatcttgt ggaaaggacg aaacaccgga gacgcgcttg tctgtaagcg
     2821 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     2881 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatta actataacgg
     2941 tcctaaggta gcgaaatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     3001 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     3061 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     3121 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggg
     3181 cgtctctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     3241 aaaaagtggc accgagtcgg tgcttttttt ctagaggtac ccgttacata acttacggta
     3301 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca
     3361 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     3421 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     3481 cccgcctggc attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     3541 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
     3601 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
     3661 gcagcgatgg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
     3721 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     3781 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     3841 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     3901 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
     3961 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
     4021 ctttttttca ggttggaccg gtgccaccat gcccaagaag aagaggaagg tgtccaatct
     4081 cctgactgtt caccagaacc tccctgcgct gccagtagat gccactagcg atgaggtcag
     4141 gaaaaatctc atggatatgt ttagggatag acaggcgttt tctgaacaca cctggaaaat
     4201 gctgcttagc gtgtgccgat cctgggcagc ctggtgtaag ctgaacaatc gcaaatggtt
     4261 ccccgccgag ccggaggacg tgcgcgatta cctgctgtat ctccaggcaa gagggctggc
     4321 tgtcaagact atccagcagc acttgggcca actgaatatg ctgcatcgac gcagcgggct
     4381 cccccggcct agcgattcaa acgcagtctc ccttgttatg aggagaatta gaaaggaaaa
     4441 cgtagatgcg ggtgagaggg ctaagcaggc tctcgctttt gagcggactg atttcgacca
     4501 ggtcagatcc ctgatggaga acagcgatcg gtgccaggac atcaggaacc tcgcatttct
     4561 gggaattgca tataacacac ttctgcgcat agctgagatc gcccggatca gagtgaaaga
     4621 catcagtcga acggacggcg gccggatgct tattcatatt ggacgcacaa agacattggt
     4681 cagcaccgct ggcgttgaaa aggccttgtc cctgggcgta acgaagctgg tggaaagatg
     4741 gatctcagtg tccggcgtgg ctgacgaccc taataattac ttgttctgtc gagtgagaaa
     4801 aaacggagtc gccgcgccct ctgccaccag ccaattgagt acacgggccc ttgaagggat
     4861 ctttgaggca acccaccgac tcatatacgg agccaaggat gacagtggcc agaggtatct
     4921 cgcctggtca ggtcattctg ctagggtggg ggccgcacga gacatggcgc gggcaggagt
     4981 ctccatacca gagattatgc aagctggagg ttggacaaat gtgaacatcg ttatgaacta
     5041 tatccgcaat cttgactctg aaaccggggc catggtgaga ctgctcgaag atggtgacta
     5101 cccatacgat gttccagatt acgctgctag cggcagtgga gagggcagag gaagtctgct
     5161 aacatgcggt gacgtcgagg agaatcctgg cccaatggac gaaaagacca caggatggcg
     5221 aggaggacac gtcgtcgagg gactggcagg agagctggaa cagctgcggg ctagactgga
     5281 acaccatcct cagggacagc gagagccagg aggaggtgga agcggaggag gaggaagcgg
     5341 aggaggaggt agcggatccg tgagcaaggg cgaggagctg ttcaccgggg tggtacccat
     5401 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     5461 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     5521 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     5581 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     5641 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     5701 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     5761 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     5821 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     5881 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     5941 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     6001 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     6061 cgagctgtac aagtccggac tcagatctcg agaggaggag gaggagacag acagcaggat
     6121 gccccacctc gacagccccg gcagctccca gcctagacgc tccttcctct caagggtgat
     6181 cagggcagcg ctaccgttgc agctgcttct gctgctgctg ctgctcctgg cctgcctgct
     6241 acctgcctct gaagatgact acagctgcac ccaggccaac aactttgccc gatccttcta
     6301 ccccatgctg cggtacacca acgggccacc tcccacctaa gcctaggaat tcgatatcaa
     6361 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     6421 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     6481 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     6541 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     6601 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     6661 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     6721 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     6781 ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     6841 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     6901 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     6961 tcgataccga gcgctaataa aagatcttta ttttcattag atctgtgtgt tggttttttg
     7021 tgtcacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     7081 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     7141 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     7201 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     7261 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     7321 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcc
//
